Vaccines comprising heat-sensitive transgenes

ABSTRACT

The present disclosure provides temperature sensitive essential nucleic acid molecules from a psychrophilic bacterium, proteins encoded by the nucleic acid molecules, as well as recombinant cells into which have been introduced such nucleic acid molecules. The disclosed recombinant cells containing one or more essential nucleic acid molecules from a psychrophilic bacterium are thereby made temperature sensitive, and can be administered to a mammal to induce an immune response in the mammal.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. application Ser. No.13/496,723 filed Apr. 12, 2012, pending, which is the U.S. NationalStage of International Application No. PCT/CA2010/001561, filed Oct. 7,2010, which was published in English under PCT Article 21(2), which inturn claims the benefit of U.S. Provisional Application No. 61/249,385filed Oct. 7, 2009, and U.S. Provisional Application No. 61/322,634filed on Apr. 9, 2010, all herein incorporated by reference.

FIELD

The technology relates to genes derived from psychrophilic bacteria, foruse in the development of heat-sensitive vaccines. In one example, thetechnology relates to recombinant pathogens harboring the heat-sensitivegene ligA from Colwellia psychrerythraea, Pseudoalteromonashaloplanktis, and Shewanella frigidimarina and to genes ligA, pyrG,hemC, ftsZ, cmk, murG, fmt, and dnaK from C. psychrerythraea.

BACKGROUND

Vaccines against bacterial and viral diseases have played an importantrole in reducing infectious diseases in humans; however, there is stilla need for innovative vaccines to reduce the current global burden ofinfectious diseases. Cold-adapted viruses have been used for decades asvaccines against human viral diseases. The best known example of such avaccine is the Sabin polio virus vaccine. An alternate example is a coldadapted influenza vaccine called FluMist® (Medimmune LLC, Gaithersburg,Md., USA), which was introduced in the U.S. in 2003. FluMist® has beenshown to be considerably more effective in certain demographic groupsthan influenza vaccines that practice the more common vaccinationstrategy of using inactivated virus to stimulate an immune response.Typically cold-adapted or “temperature-sensitive” (TS) viral strainshave been developed by passing the virus repeatedly in eggs or cellculture at low temperatures and then testing the progeny for theirinability to grow above about 37° C., generally thought of as the“normal” human body temperature.

The concept of a “normal” human body temperature takes intoconsideration anatomical sites, individual variations, gender,physiological conditions and ambient temperature. Despite the number ofvariables, the human body can function only in a very narrow temperaturerange, which is generally about 36° C.-39° C. If the human body coretemperature falls to about 35° C., the body must be warmed or death willensue. The skin temperature is always cooler than the body coreregardless of the ambient temperature and clothing worn. At moderatetemperatures (e.g., 21° C.), the temperature of the skin is about 32°C.-35° C.

Those skilled in these arts are of the view that bacteria generally havea set of about 100 to 150 genes, called “essential genes” that areabsolutely required for maintenance of bacterial viability. Identifyingessential genes is difficult due to their nature, as knockouts of thesegenes results in death of the organism. Essential genes encode proteinscomposed of amino acid sequences that are highly conserved among almostall bacterial genera and species. This conservation presumably reflectstheir common function and structure among the different species. Aselect number of essential genes have been shown to be competent insubstituting for a homologue in another bacterial species and in somecases these substitutions were from distantly related bacterial species.The conservation of amino acid sequences is widespread among bacteria,the deduced amino acid sequences of essential genes from psychrophilesand thermophiles shows high identity with their mesophilic counterparts.Microbiologists have generally used conditional lethal mutations, suchas TS mutations, to identify essential genes.

Many bacterial species play significant roles in the global burden ofinfectious diseases. However, the causative agent of tuberculosis isprobably the most significant contributor to human morbidity andmortality caused by an infectious bacterial disease. Although theBacille Calmette-Guérin (BCG) vaccine has been used for several decadesto protect against tuberculosis, its low efficacy has failed to lowerthe incidence of tuberculosis to acceptable levels.

SUMMARY

The present disclosure provides methods for engineering, producing andusing heat-sensitive host microbial cells. In one example, recombinantpathogens contain heat-sensitive essential genes, for example insertedusing homologous recombination. “Psychrophile” is a term that is appliedto organisms that function optimally at cold temperatures e.g., <20° C.Bacteria that live in cold ocean water, especially the Arctic andAntarctic oceans, are examples of psychrophilic bacteria. Enzymes andother proteins in psychrophilic bacteria function better in the coldthan their homologous counterparts in mesophilic bacteria. Many of theenzymes from psychrophilic bacteria are also prone to denaturation attemperatures much lower than those that would affect their mesophiliccounterpart. Presumably the pattern of temperature-sensitivity ofpsychrophilic enzymes extends to the products of essential genes.

Methods of identifying and manipulating psychrophilic essential geneswith desired TS properties are provided. in vitro and in vivorecombinant technologies can be used. Francisella tularensis is theetiologic agent of the zoonotic disease, tularaemia. It can infectnumerous animals by a variety of routes, and typically infects and growsin monocyte-derived cells in organs of the reticuloendothelial system. Aclosely related bacterium, Francisella novicida, has many of theproperties of F. tularensis, and, in addition, is highly amenable tomany genetic manipulations, including gene substitutions. Thepathophysiology and genetic properties of F. novicida make it ideal forstudying the effects of gene substitutions on a pathogenic bacterium. F.novicida is a mesophile with a maximal growth temperature of about 45°C.

This disclosure also provides methods to determine maximal growthtemperature of both bacterial strains and their growth properties atrestrictive temperatures. The recombinant bacterial strains tested grewbelow the restrictive temperature but not above the restrictivetemperature. When a psychrophilic essential allele encoding an essentialgene is inserted into an area of a mammalian body that is colder thanthe human body core, e.g., the skin, the recombinant pathogenic bacteriawill have the ability to thrive thereby inducing an immune response.When the pathogenic recombinant bacteria migrate to organs in the humanbody core where the temperature is higher, they die and are unable toharm the host.

The present disclosure provides isolated temperature-sensitive essentialnucleic acid molecules from a psychrophilic bacterium comprising atleast 80%, at least 90%, or at least 95% sequence identity to thenucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 22, 23, or 24. In some examples, the psychrophilic bacteria areoperable at a temperature of about −10° C. to about 30° C., butinoperable at a temperature greater than about 30° C. Vectors andrecombinant host cells (such as a recombinant bacterial host cell) thatinclude such temperature-sensitive essential nucleic acid molecules froma psychrophilic bacterium are also provided. Immunogenic compositionsthat include such recombinant host bacteria (such as live or killedcells) are also disclosed. The disclosure also provides isolatedproteins encoded by the disclosed isolated temperature-sensitiveessential nucleic acid molecules, such as proteins having at least 80%,at least 90%, or at least 95% sequence identity to the amino acidsequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 26,27, or 28.

Methods of making a temperature-sensitive microbial host cell, such as arecombinant host cell, are provided. In one example the method includesintroducing (for example by inserting, substituting or replacing) anucleic acid construct into the genome of a mesophilic bacterial strain,wherein the nucleic acid construct includes a temperature-sensitiveessential nucleic acid molecule from a psychrophilic bacterial strainand one or more control sequences operably linked to thetemperature-sensitive essential nucleic acid molecule, wherein thetemperature-sensitive essential peptide encoded by the introducedtemperature-sensitive essential nucleic acid molecule is operable (e.g.,functional) at a temperature less than about 30° C. and inoperable(e.g., non-functional) at a temperature greater than about 30° C. Insome examples the method also includes culturing thetemperature-sensitive microbial host cell at a temperature wherein thetemperature-sensitive peptide is operable, whereby said microbial hostcell produces a plurality of peptides; increasing the culturingtemperature to a temperature at which the temperature-sensitive peptideis inoperable; maintaining said culturing for a period of timesufficient to kill the temperature-sensitive microbial host cell; andharvesting the killed temperature-sensitive microbial host cells.

Methods for producing an immune response to a bacterium in a subjectusing the disclosed nucleic acid molecules, proteins, and recombinanthost cells are provided. In one example the method includesadministering to the subject a therapeutically effective amount of atemperature-sensitive bacterium, wherein the temperature-sensitivebacterium expresses a temperature-sensitive essential nucleic acidmolecule from a psychrophilic bacterial strain, thereby inducing animmune response to the bacterium. Such methods can be used to prevent ortreat a bacterial infection (such as a M. tuberculosis, Salmonella orFrancisella infection).

The foregoing and other features of the disclosure will become moreapparent from the following detailed description of several embodimentswhich proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a flowchart illustrating an exemplary method using polymerasechain reaction (PCR), FIG. 1b is a schematic chart illustrating anexemplary method showing DNA integration-excision events that result ina gene substitution.

FIG. 2a is a schematic illustrating the sequence of the wild type (wt)F. novicida ligA gene as it exists normally in a chromosome, FIG. 2b isa schematic illustrating ligA_(Cp) gene substitutions into the F.novicida chromosome according to an exemplary method of the presentdisclosure, FIG. 2c is a schematic illustrating ligA_(Sf) genesubstitution into the F. novicida chromosome according to an exemplarymethod of the present disclosure, FIG. 2d is a schematic illustratingligA_(Ph) gene substitutions into the F. novicida chromosome accordingto an exemplary method of the present disclosure, FIG. 2e is a schematicillustrating ligA_(Ph2) gene substitutions into the F. novicidachromosome according to an exemplary method of the present disclosure.

FIG. 3a is a graph illustrating the growth curve of wt F. novicida andF. novicida with the C. psychrerythraea ligA_(Cp) gene substituted forthe F. novicida homologue at 30° C., FIG. 3b is a graph illustrating thegrowth curve of F. novicida with the C. psychrerythraea ligA_(Cp) genesubstituted for the F. novicida homologue and wt F. novicida with atemperature shift from 30° C. to 33° C. after 2 hours, FIG. 3c is agraph illustrating the growth curve of F. novicida with the C.psychrerythraea ligA_(Cp) gene substituted for the F. novicida homologueand wt F. novicida with a temperature shift from 30° C. to 34° C. after3.5 hours, FIG. 3d is a graph illustrating the growth curve of F.novicida with the C. psychrerythraea ligA_(Cp) gene substituted for theF. novicida homologue and wt F. novicida with a temperature shift from30° C. to 35° C. after 2 hours, FIG. 3e is a graph illustrating thegrowth curve of F. novicida with the C. psychrerythraea ligA_(Cp) genesubstituted for the F. novicida homologue and wt F. novicida with atemperature shift from 30° C. to 37° C. after 2 hours.

FIG. 4a is a graph illustrating the growth curve of wt F. novicida andF. novicida with the S. frigidimarina ligA_(Sf) gene substituted for theF. novicida homologue at 30° C., FIG. 4b is a graph illustrating thegrowth curve of F. novicida with the S. frigidimarina ligA_(Sf) genesubstituted for the F. novicida homologue and wt F. novicida with atemperature shift from 30° C. to 33° C. after 2 hours, FIG. 4c is agraph illustrating the growth curve of F. novicida with the S.frigidimarina ligA_(Sf) gene substituted for the F. novicida homologueand wt F. novicida with a temperature shift from 30° C. to 35° C. after2 hours, FIG. 4d is a graph illustrating the growth curve of F. novicidawith the S. frigidimarina ligA_(Sf) gene substituted for the F. novicidahomologue and wt F. novicida with a temperature shift from 30° C. to 37°C. after 2 hours.

FIG. 5a is a graph illustrating the growth curve of wt F. novicida andF. novicida with the P. haloplanktis ligA_(Ph) gene substituted for theF. novicida homologue at 30° C., FIG. 5b is a graph illustrating thegrowth curve of F. novicida with the P. haloplanktis ligA_(Ph) genesubstituted for the F. novicida homologue and wt F. novicida with atemperature shift from 30° C. to 33° C. after 2 hours, FIG. 5c is agraph illustrating the growth curve of F. novicida with the P.haloplanktis ligA_(Ph) gene substituted for the F. novicida homologueand wt F. novicida with a temperature shift from 30° C. to 35° C. after2 hours, FIG. 5d is a graph illustrating the growth curve of F. novicidawith the P. haloplanktis ligA_(Ph) gene substituted for the F. novicidahomologue and wt F. novicida with a temperature shift from 30° C. to 37°C. after 2 hours.

FIG. 6a is a graph illustrating the growth curve of wt F. novicida andF. novicida with the P. haloplanktis ligA_(Ph2) gene substituted for theF. novicida homologue at 21° C., FIG. 6b is a graph illustrating thegrowth curve of F. novicida with the P. haloplanktis ligA_(Ph2) genesubstituted for the F. novicida homologue and wt F. novicida with atemperature shift from 21° C. to 26° C. after 2 hours, FIG. 6c is agraph illustrating the growth curve of F. novicida with the P.haloplanktis ligA_(Ph2) gene substituted for the F. novicida homologueand wt F. novicida with a temperature shift from 21° C. to 28° C. after2 hours, FIG. 6d is a graph illustrating the growth curve of F. novicidawith the P. haloplanktis ligA_(Ph2) gene substituted for the F. novicidahomologue and wt F. novicida with a temperature shift from 21° C. to 30°C. after 2 hours.

FIG. 7 is a graph illustrating the decline in viability of wt F.novicida and F. novicida—ligA_(Cp) cultures at 37° C. after being grownto late exponential phase at 33° C.

FIG. 8 is a digital image illustrating the growth of S. ser.Typhimurium-ligA_(CP) at 30° C. and the lack of growth at 37° C.

FIG. 9a is a graph illustrating the growth curve of wt Mycobacteriumsmegmatis and M. smegmatis-ligA_(Cp) at 30° C., FIG. 9b is a graphillustrating the growth curve of M. smegmatis-ligA_(CP) and wt M.smegmatis with a temperature shift from 30° C. to 35° C. after 4 hours,FIG. 9c is a graph illustrating the growth curve of M.smegmatis-ligA_(CP) and wt M. smegmatis with a temperature shift from30° C. to 37° C. after 4 hours.

FIGS. 10a-10d are a series of graphs showing the protective immunityinduced by TS F. novicida strains.

FIGS. 11A-11L show sequences disclosed herein, with underlined portionsbeing F. novicida sequence.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood to be included by any reference to the displayed strand.

SEQ ID NO: 1 is a full length nucleic acid coding sequence of theligA_(Cp) hybrid gene.

SEQ ID NO: 2 is the deduced 689 amino acid sequence of LigA_(Cp) hybridprotein.

SEQ ID NO: 3 is a full length nucleic acid coding sequence of theligA_(Ph) hybrid gene.

SEQ ID NO: 4 is the deduced 673 amino acid sequence of LigA_(Ph) hybridprotein.

SEQ ID NO: 5 is a full length nucleic acid coding sequence of theligA_(Ph2) hybrid gene.

SEQ ID NO: 6 is the deduced 673 amino acid sequence of LigA_(Ph2) hybridprotein.

SEQ ID NO: 7 is a full length nucleic acid coding sequence of theligA_(Sf) hybrid gene.

SEQ ID NO: 8 is the deduced 670 amino acid sequence of LigA_(Sf) hybridprotein.

SEQ ID NO: 9 is a full length nucleic acid coding sequence of thepyrG_(Cp) hybrid gene.

SEQ ID NO: 10 is the deduced 545 amino acid sequence of PyrG_(Cp) hybridprotein.

SEQ ID NO: 11 is a full length nucleic acid coding sequence of thehemC_(Cp) hybrid gene.

SEQ ID NO: 12 is the deduced 317 amino acid sequence of HemC_(Cp) hybridprotein.

SEQ ID NO: 13 is a full length nucleic acid coding sequence of thefmt_(Cp) hybrid gene.

SEQ ID NO: 14 is the deduced 327 amino acid sequence of Fmt_(Cp) hybridprotein.

SEQ ID NO: 15 is a full length nucleic acid coding sequence of themurG_(Cp) hybrid gene.

SEQ ID NO: 16 is the deduced 387 amino acid sequence of MurG_(Cp) hybridprotein.

SEQ ID NO: 17 is a full length nucleic acid coding sequence of codonoptimized ligA_(Cp) optimized for M. tuberculosis.

SEQ ID NO: 18 is the deduced 689 amino acid coding sequence of codonoptimized LigA_(Cp) hybrid protein with the first four codons changed tothe M. tuberculosis form.

SEQ ID NO: 19 is a full length nucleic acid coding sequence of thednaK_(Cp) hybrid gene.

SEQ ID NO: 20 is the deduced 638 amino acid coding sequence of DnaK_(Cp)hybrid protein.

SEQ ID NOS: 21 and 22 are a full length nucleic acid coding sequence ofthe essential gene tyrS from Colwellia psychrerythraea, and thecorresponding amino acid sequence, respectively.

SEQ ID NO: 23 and 24 are a full length nucleic acid coding sequence ofthe essential gene cmk from Colwellia psychrerythraea and thecorresponding amino acid sequence, respectively. As shown in FIG. 11J,F. novicida sequence is underlined. The underlined regions correspond tothe F. novicida sequence in both the nucleotide and amino acid sequence.The “non-underlined” is Colwellia psychrerythraea sequence. In the aminoacid sequence there is no underlined amino acids at the end since the F.novicida sequence starts at the stop codon.

SEQ ID NO: 25 and 26 are a full length nucleic acid coding sequence ofthe essential gene dnaKsf from Shewanella frigidimarina and thecorresponding amino acid sequence, respectively. As shown in FIG. 11K,Francisella novicida sequence is underlined. The underlined regionscorrespond to the F. novicida sequence in both the nucleotide and aminoacid sequence. The “non-underlined” shows the Shewanella frigidimarinasequence. In the amino acid sequence at the beginning (MGK) is identicalbetween Shewanella and Francisella, so it is double underlined. Thesingle underline at the end of the amino acid sequence corresponds tothe F. novicida sequence.

SEQ ID NO: 27 and 28 are a full length nucleic acid coding sequence ofthe essential gene ftsZ from Colwellia psychrerythraea and thecorresponding amino acid sequence, respectively. As shown in FIG. 11L,Francisella novicida sequence is underlined. The underlined regionscorrespond to the F. novicida sequence in both the nucleotide and aminoacid sequence. The “non-underlined” regions are Colwelliapsychrerythraea sequence. There is extensive F. novicida region at the5′-end (N-terminus).

DETAILED DESCRIPTION

The following explanations of terms and methods are provided to betterdescribe the present disclosure. The singular forms “a,” “an,” and “the”refer to one or more than one, unless the context clearly dictatesotherwise. For example, the term “comprising a nucleic acid molecule”includes single or plural nucleic acid molecules and is consideredequivalent to the phrase “comprising at least one nucleic acidmolecule.” The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise. As used herein, “comprises”means “includes.” Thus, “comprising A or B,” means “including A, B, or Aand B,” without excluding additional elements.

Suitable methods and materials for the practice and/or testing ofembodiments of the disclosure are described below. Such methods andmaterials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein can be used. For example, conventional methods well known in theart to which a disclosed invention pertains are described in variousgeneral and more specific references, including, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel etal., Current Protocols in Molecular Biology, Greene PublishingAssociates, 1992 (and Supplements to 2000); Ausubel et al., ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1999.

The references cited herein are incorporated by reference.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided.Unless otherwise noted, technical terms are used according toconventional usage by those skilled in the arts

Adjuvant: A vehicle used to enhance antigenicity, for exampleantigenicity of a recombinant host bacterium containing a TS essentialpsychrophilic bacteria sequence disclosed herein. Adjuvants include asuspension of minerals (e.g., alum, aluminum hydroxide, or phosphate) onwhich antigen is adsorbed; or water-in-oil emulsion in which antigensolution is emulsified in mineral oil (Freund incomplete adjuvant),sometimes with the inclusion of killed mycobacteria (Freund's completeadjuvant) to further enhance antigenicity (inhibits degradation ofantigen and/or causes influx of macrophages). Immunostimulatoryoligonucleotides (such as those including a CpG motif) can also be usedas adjuvants (for example see U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199).Adjuvants include biological molecules (a “biological adjuvant”), suchas costimulatory molecules. Exemplary adjuvants include IL-2, RANTES,GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

Administration: The introduction of a composition (such as animmunogenic composition) into a subject (such as a mammal, for example ahuman) by a selected route. Exemplary routes of administration include,but are not limited to, topical, injection (such as subcutaneous,intramuscular, intradermal, intraperitoneal, intratumoral, andintravenous), oral, sublingual, rectal, transdermal, intranasal, vaginaland inhalation routes.

Ameliorate: The improvement of a disease or pathological condition (suchas a bacterial infection) with respect to the effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease in a susceptible subject, areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease, an improvement in the overall healthor well-being of the subject, or by other parameters well known to thoseskilled in the arts specific to the particular disease.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes mammals and birds. The term “mammal” includes both human andnon-human mammals. Similarly, the term “subject” includes both human andveterinary subjects (such as mice, rats, rabbits, dogs, cats, horses,and cattle).

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen. Antibodies are composed of a heavyand a light chain, each of which has a variable region, termed thevariable heavy (V_(H)) region and the variable light (V_(L)) region.Together, the V_(H) region and the V_(L) region are responsible forbinding the antigen recognized by the antibody.

Antibodies include intact immunoglobulins and the variants and portionsof antibodies well known in the art, such as Fab fragments, Fab′fragments, F(ab)′₂ fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

“Specifically binds” refers to the ability of individual antibodies tospecifically immunoreact with an antigen, such as a bacterial antigen,relative to binding to unrelated proteins, such as non-bacterialproteins. The binding is a non-random binding reaction between anantibody molecule and an antigenic determinant of the T cell surfacemolecule. The desired binding specificity is typically determined fromthe reference point of the ability of the antibody to differentiallybind the T cell surface molecule and an unrelated antigen, and thereforedistinguish between two different antigens, particularly where the twoantigens have unique epitopes. An antibody that specifically binds to aparticular epitope is referred to as a “specific antibody”.

In some examples, an antibody specifically binds to a target (such as abacterial protein) with a binding constant that is at least 10³ M⁻¹greater, 10⁴ M⁻¹ greater or 10⁵ M⁻¹ greater than a binding constant forother molecules in a sample or subject. In some examples, an antibody orfragments thereof, has an equilibrium constant (Kd) of 1 nM or less. Forexample, an antibody binds to a target, such as a bacterial protein witha binding affinity of at least about 0.1×10⁻⁸M, at least about 0.3×10⁻⁸M, at least about 0.5×10⁻⁸ M, at least about 0.75×10⁻⁸ M, at least about1.0×10⁻⁸ M, at least about 1.3×10⁻⁸ Mat least about 1.5×10⁻⁸M, or atleast about 2.0×10⁻⁸ M. Kd values can, for example, be determined bycompetitive ELISA (enzyme-linked immunosorbent assay) or using asurface-plasmon resonance device such as the Biacore T100, which isavailable from Biacore, Inc., Piscataway, N.J.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Generally, T cellsrecognize epitopes of continuous amino acids. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance.

Examples of antigens include, but are not limited to, peptides, lipids,polysaccharides, and nucleic acids containing antigenic determinants,such as those recognized by an immune cell. An antigen can be atissue-specific antigen, or a disease-specific antigen. These terms arenot exclusive, as a tissue-specific antigen can also be a diseasespecific antigen. A tissue-specific antigen is expressed in a limitednumber of tissues, such as a single tissue. A tissue specific antigenmay be expressed by more than one related type of tissue, such asalveolar and bronchial tissue. A disease-specific antigen is expressedcoincidentally with a disease process. Specific non-limiting examples ofa disease-specific antigen are an antigen whose expression correlateswith, or is predictive of, a bacterial infection, such as tuberculosis.A disease-specific antigen can be an antigen recognized by T cells or Bcells.

CD4: Cluster of differentiation factor 4, a T cell surface protein thatmediates interaction with the MHC Class II molecule. CD4 also serves asthe primary receptor site for HIV on T cells during HIV infection. Cellsthat express CD4 are often helper T cells.

CD8: Cluster of differentiation factor 8, a T cell surface protein thatmediates interaction with the MHC Class I molecule. Cells that expressCD8 are often cytotoxic T cells. “CD8+ T cell mediated immunity” is animmune response implemented by presentation of antigens to CD8+ T cells.

Contacting: The process of incubating one agent in the presence ofanother. Thus, when a cell is contacted with an agent (such as animmunogenic composition), the cell is incubated with the agent for asufficient period of time for the agent and the cell to interact.

Cool parts of the body: Regions of a human or other mammalian body thatgenerally have a lower temperature than other parts of the body. Theconcept of natural human (or other mammal) body temperature variationdue to anatomical sites, gender, physiological and ambient temperature.Despite the number of variables, the human (or other mammalian) body canfunction only in a very narrow temperature range, hence, for example thehuman body core remains at about 36° C.-39° C. Cool parts of the bodyinclude skin, mouth and rectum. Skin temperature, for example, is about32° C.-35° C. Thus, in some examples, cool parts of the body havetemperatures that are at least 1° C. less, at least 2° C. less, at least3° C. less, at least 4° C. less, at least 4° C. less, or at least 6° C.less, such as 1° C. to 8° C. less, 1° C. to 6° C. less, 2° C. to 6° C.less, or 2° C. to 4° C. less, than other parts of the body, such as thecore.

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking. Specific,non-limiting examples of cytokines include the interleukins (IL-2, IL-4,IL-6, IL-10, IL-21, etc.), and IFN-γ.

Degenerate variant: A TS essential psychrophilic bacteria nucleic acidsequence that encodes a TS essential psychrophilic bacteria protein thatincludes a nucleic acid sequence that is degenerate as a result of thegenetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in this disclosure as long as the amino acidsequence of the TS essential psychrophilic bacteria peptide encoded bythe nucleotide sequence is unchanged.

Em^(R): Erythromycin resistance.

Essential gene: A gene that is necessary for the growth of the organism(such as a mesophilic bacterium) under all culturing conditions.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters, are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences. In one embodiment, thepromoter is a cytomegalovirus promoter.

Heat-sensitive: An inability to perform an essential biological functionat temperatures above about 28° C. Similarly, the term “heat-sensitiveprotein or polypeptide” refers to a non-functional mature proteinresulting from heat-induced deactivation. An enzyme that does notcatalyze its known reaction efficiently enough to support growth,development or life of the organism above about 28° C. is an example ofsuch a protein.

Heat-sensitive allele: An allele comprising a gene encoding aheat-sensitive protein. Similarly the term, “heat-sensitive gene” refersto a gene encoding a heat-sensitive protein.

Host cells: Cells into which a heterologous nucleic acid molecule hasbeen introduced. For example, such cells may include a nucleic acidvector that is propagated and its DNA expressed. The cell may beprokaryotic or eukaryotic. The cell can be prokaryotic, such as abacterial cell. The term also includes any progeny of the subject hostcell. It is understood that all progeny may not be identical to theparental cell since there may be mutations that occur duringreplication. However, such progeny are included when the term “hostcell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen or a particular TSrecombinant microbial cell, such as mesophilic bacteria containing apsychrophile essential nucleic acid molecule provided herein. In oneembodiment, an immune response is a T cell response, such as a CD4+response or a CD8+ response. In another embodiment, the response is a Bcell response, and results in the production of specific antibodies. Thedevelopment of an immune response following administration of mesophilicbacteria containing a psychrophile TS essential nucleic acid moleculecan be measured using routine methods known in the art, for example bymeasuring cytokine production as an indication of a protective immuneresponse.

Immunogenic composition: Compositions that include recombinantmesophilic bacteria containing a psychrophile TS essential nucleic acidmolecule that induces a measurable CTL response against a recombinantmesophilic bacteria protein, or induces a measurable B cell response(such as production of antibodies that specifically bind a recombinantmesophilic bacteria-specific protein) against a recombinant mesophilicbacteria protein. For example, the immunogenic polypeptide or a nucleicacid encoding the immunogenic polypeptide can be present in aheat-sensitive mesophilic bacteria generated using the methods providedherein, wherein the bacteria is art of an immunogenic composition thatcan further include pharmaceutically acceptable carriers, and/or othertherapeutic agents. An immunogenic composition can optionally include anadjuvant, a PD-1 antagonist, a co-stimulatory molecule, or a nucleicacid encoding a costimulatory molecule. An immunogenic composition canbe readily tested for its ability to induce a CTL by art-recognizedassays.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response(e.g. antibody production) against the antigen from which theimmunogenic peptide is derived. Immunogenic peptides can also beidentified by measuring their binding to a specific MHC protein and bytheir ability to stimulate CD4 and/or CD8 when presented in the contextof the MHC protein.

Generally, immunogenic polypeptides can be used to induce an immuneresponse in a subject, such as a B cell response or a T cell response.In one example, an immunogenic polypeptide, when bound to a MHC Class Imolecule, activates cytotoxic T lymphocytes (CTLs) against thepolypeptide. Induction of CTLs using synthetic peptides and CTLcytotoxicity assays are known in the art, see U.S. Pat. No. 5,662,907.In one example, an immunogenic peptide includes an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response against the antigenfrom which the immunogenic peptide is derived.

Immunologically reactive conditions: Conditions that allow an antibodyspecific for a particular epitope to bind to that epitope to a greaterdegree than, and/or to the substantial exclusion of, binding tosubstantially all other epitopes. These conditions are dependent uponthe format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols or those conditions encountered invivo. The immunologically reactive conditions employed in the disclosedmethods are “physiological conditions” which include reference toconditions (e.g., temperature, osmolarity, pH) that are typical inside aliving mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organ andintracellular environment is generally about pH 7 (e.g., from pH 6.0 topH 8.0, or pH 6.5 to pH 7.5, such as pH 7.2), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation. These conditions are well known to thoseskilled in these arts.

Interferon gamma (IFN-γ): IFN-γ is a dimeric protein with subunits of146 amino acids. The protein is glycosylated at two sites, and the pI is8.3-8.5. IFN-γ is synthesized as a precursor protein of 166 amino acidsincluding a secretory signal sequence of 23 amino acids. Two molecularforms of the biologically active protein of 20 and 25 kDa have beendescribed. Both of them are glycosylated at position 25. The 25 kDa formis also glycosylated at position 97. The observed differences of naturalIFN-γ with respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kDa forms observed under non-denaturingconditions are dimers and tetramers of IFN-γ. The human gene has alength of approximately 6 kb. It contains four exons and maps tochromosome 12q24.1.

IFN-γ can be detected by sensitive immunoassays, such as an ELISPOT testthat allows detection of individual cells producing IFN-γ. Minuteamounts of IFN-γ can be detected indirectly by measuring IFN-inducedproteins such as Mx protein. The induction of the synthesis of IP-10 hasbeen used also to measure IFN-γ concentrations. In addition, bioassayscan be used to detect IFN-γ, such as an assay that employs induction ofindoleamine 2,3-dioxygenase activity in 2D9 cells. The production ofIFN-γ can be used to assess T cell activation, such as activation of a Tcell by bacterial antigen.

Isolated: A biological component (such as a nucleic acid molecule,protein or organelle) that has been substantially separated or purifiedaway from other biological components in the cell of the organism inwhich the component naturally occurs, e.g., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidmolecules and proteins that have been “isolated” include nucleic acidmolecules and proteins purified by standard purification methods. Inanother embodiment, “isolated” refers to nucleic acid molecules andproteins prepared by recombinant expression in a host cell as well aschemically synthesized nucleic acids.

ligA: A wt allele of the gene encoding NAD-dependent DNA ligase found inmesophilic bacteria such as F. novicida, M. smegmatis or E. coli.Furthermore, ligA with a subscript, such as ligA_(Cp), ligA_(Sf), orligA_(Ph), refers to a wt allele of the gene encoding NAD-dependent DNAligase found in psychrophilic bacteria. For example ligA_(Cp) refers tothe wt allele of ligA found in the Arctic bacterium C. psychrerythraeastrain 34H which has a maximal growth temperature below 18° C. The ligAsequences from psychrophilic bacteria can be introduced into mesophilicbacteria, to confer temperature sensitivity to the mesophilic bacteria.

Mesophile: An organism naturally found in environments at temperaturesbetween about 20° C. and 50° C. A bacterial mesophile refers to abacterium that is normally associated with a mammal and thus is normallyfunctioning at temperatures between about 32° C. and 45° C.

Psychrophile: An organism naturally found in environments that arepermanently below 20° C., often permanently below 10° C. and sometimesbelow 0° C. Such permanently cold environments include most oceanenvironments, permafrost soils, Arctic and Antarctic environments. Thoseskilled in these arts will understand that “psychrophile” and“psychrotroph” are commonly used to describe bacteria that grow in coldenvironments.

Psychrophilic: Features found in psychrophiles. For example, a“psychrophilic enzyme” is an enzyme isolated from a psychrophile.

Peptide modifications: Analogs (non-peptide organic molecules),derivatives (chemically functionalized peptide molecules obtainedstarting with the disclosed peptide sequences) and variants (homologs)of proteins that can be used in the methods and compositions providedherein. Peptides are comprised of amino acids, which may be either L-and/or D-amino acids, naturally occurring and otherwise. The peptidescan be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties.Modifications are well known to those skilled in these arts.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic composition, such as an immunogenic composition.

The disclosed purified active compositions can be administered alone orcombined with an acceptable carrier. Preparations can contain one typeof therapeutic molecule, or can be composed of a combination of severaltypes of therapeutic molecules. The nature of the carrier will depend onthe particular mode of administration being utilized.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to be at risk of infection with M. tuberculosis, or M.leprae. An example of a person with a known predisposition is someoneliving with a person diagnosed with tuberculosis, health careprofessionals, or someone otherwise known to have been exposed to M.tuberculosis. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological condition,such as tuberculosis, after it has begun to develop.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein is more pure than the protein inits originating environment within a cell. A preparation of a protein istypically purified such that the protein represents at least 50% of thetotal protein content of the preparation. However, more highly purifiedpreparations may be required for certain applications. For example, forsuch applications, preparations in which the protein includes at least75% or at least 90% of the total protein content may be employed.

Recombinant: A nucleic acid molecule that has a sequence not naturallyoccurring or a sequence that is made by an artificial combination of twonaturally separated segments of sequence. This artificial combination isoften accomplished by chemical synthesis or by the artificialmanipulation of isolated segments of nucleic acids, by geneticengineering techniques, for example. Also refers to cells into which anon-native nucleic acid molecule has been introduced.

Resistant to infection: Animals (e.g., mammals) that demonstratedecreased symptoms of infection compared to non-resistant animals.Evidence of resistance to infection can appear as, for example, lowerrates of mortality, increased life spans measured after exposure to theinfective agent, fewer or less intense physiological symptoms, such asfewer lesions, or decreased cellular or tissue concentrations of theinfective agent. In one embodiment, resistance to infection isdemonstrated by a heightened immune response.

Restrictive temperature: The lowest temperature at which an organism isunable to grow. For example, in Table 1 “restrictive temperature”specifically refers to the lowest temperature at which the F. novicidastrain with a psychrophilic gene integrated is unable to form anisolated colony on agar media. Due to the variation in the temperatureof incubators, these temperatures are interpreted as being about ±1° C.

sacB cassette: A modular DNA sequence encoding the enzyme levansucrasefrom Bacillus subtilus. Expression of this gene is lethal in thepresence of sucrose to many bacteria and can thus be used as acounter-selection agent to help select for the loss of gene sequences.

Selective hybridization: Hybridization under moderately or highlystringent conditions that exclude non-related nucleotide sequences, thetechniques of hybridization are known to those skilled in these arts.

Sequence identity: The identity/similarity between two or more nucleicacid sequences, or two or more amino acid sequences, expressed in termsof the identity or similarity between the sequences. Sequence identitycan be measured in terms of percentage identity; the higher thepercentage, the more identical the sequences are. Sequence similaritycan be measured in terms of percentage similarity (which takes intoaccount conservative amino acid substitutions); the higher thepercentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. If the two compared sequences sharehomology, then the designated output file will present those regions ofhomology as aligned sequences. If the two compared sequences do notshare homology, then the designated output file will not present alignedsequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1154 nucleotidesis 75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 15-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(that is, 15÷20*100=75).

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). Homologs are typically characterizedby possession of at least 30% sequence identity or more counted over thefull-length alignment with an amino acid sequence using the NCBI BasicBlast 2.0, gapped blastp with databases such as the nr or swissprotdatabase. Queries searched with the blastn program are filtered withDUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).Other programs use SEG. In addition, a manual alignment can beperformed. Proteins with even greater similarity will show increasingpercentage identities when assessed by this method, such as at leastabout 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with aprotein disclosed herein. Thus in one example, a protein that can beused in the disclosed methods and compositions has at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26 and 28 and retains the ability to confer TS (such asheat-sensitivity) to a mesophilic bacteria.

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions, as described above. Nucleic acid sequences that do not showa high degree of identity may nevertheless encode identical or similar(conserved) amino acid sequences, due to the degeneracy of the geneticcode. Changes in a nucleic acid sequence can be made using thisdegeneracy to produce multiple nucleic acid molecules that all encodesubstantially the same protein. Such homologous nucleic acid sequencescan, for example, possess at least about 60%, 70%, 80%, 90%, 95%, 98%,or 99% sequence identity with a disclosed nucleic acid sequence asdetermined by this method. Thus in one example, a nucleic acid sequencethat can be used in disclosed methods and compositions has at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, orat least 99% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, and 27 and retains the ability to encode a proteinthat can confer TS (such as heat-sensitivity) to a mesophilic bacteria.An alternative (and not necessarily cumulative) indication that twonucleic acid sequences are substantially identical is that the peptidewhich the first nucleic acid encodes is immunologically cross reactivewith the peptide encoded by the second nucleic acid.

Temperature-sensitive (TS)” or “heat-sensitive (HS): A bacterialcomponent (such as a protein) or bacterium that is active up to about30° C. and inactivated at a temperature that is normally found in thehuman body, e.g., above about 30° C.

Tester strain: A mesophilic bacterium that is amenable to genereplacement allowing the substitution of a psychrophilic essential genefor the homologue naturally found in the tester strain.

Therapeutically effective amount: An amount of a composition that alone,or together with an additional therapeutic agent(s) sufficient toachieve a desired effect in a subject, or in a cell, being treated withthe agent. The effective amount of the agent (such as an immunogeniccomposition provided herein) can be dependent on several factors,including, but not limited to the subject or cells being treated, theparticular therapeutic agent, and the manner of administration of thetherapeutic composition. In one example, a therapeutically effectiveamount or concentration is one that is sufficient to preventadvancement, delay progression, or to cause regression of a disease, orwhich is capable of reducing symptoms caused by the disease, such as abacterial infection (e.g., tuberculosis).

In one example, a desired response is to reduce or inhibit one or moresymptoms associated with a bacterial infection. The one or more symptomsdo not have to be completely eliminated for the composition to beeffective. The effective amount of an agent that includes one of thedisclosed immunogenic compositions that is administered to a human orveterinary subject will vary depending upon a number of factorsassociated with that subject, for example the overall health of thesubject. An effective amount of an agent can be determined by varyingthe dosage of the product and measuring the resulting therapeuticresponse, such as the prevention of bacterial infection. Effectiveamounts also can be determined through various in vitro, in vivo or insitu immunoassays. The disclosed agents can be administered in a singledose, or in several doses, as needed to obtain the desired response.

In particular examples, a therapeutically effective dose of animmunogenic composition includes at least 10² colony forming units(CFU), such as at least 10³, at least 10⁴, at least 10⁵, at least 10⁶,at least 10⁷, or at least 10⁸ CFU, for example 10² to 10⁸ CFU. In oneexample, 10² to 10⁸ CFU of live bacteria are administered intradermallyor intranasally. However, one skilled in the art will recognize thathigher or lower dosages also could be used, for example depending on theparticular immunogenic composition. In particular examples, such dailydosages are administered in one or more divided doses (such as 2, 3, or4 doses) or in a single formulation. The disclosed immunogeniccomposition can be administered alone, in the presence of apharmaceutically acceptable carrier, in the presence of othertherapeutic agents.

Treatment: A therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop. Inone example, the immunogenic compositions disclosed herein followingadministration to a mammal achieves a reduction in one or more signs ofa bacterial infection.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transduced or transformed host cell, referred to herein as arecombinant cell. A vector may include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector may also include one or more selectable marker gene and othergenetic elements known in the art. Vectors include plasmid vectors,including plasmids for expression in gram negative and gram positivebacterial cells. Exemplary vectors include those for expression in E.coli and Salmonella. Vectors also include viral vectors, such as, butare not limited to, retrovirus, orthopox, avipox, fowlpox, capripox,suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbisvirus, vaccinia virus and poliovirus vectors.

Temperature-Sensitive Essential Genes from Psychrophilic Bacteria

It is disclosed herein that several nucleic acid molecules, and theircorresponding peptides, can be introduced into a bacteria to confertemperature sensitivity (TS), such as heat-sensitivity, to the hostbacteria. The resulting bacteria can be used to induce an immuneresponse to the temperature-sensitive bacteria, such as a T cellresponse. Exemplary psychrophilic essential genes with desiredtemperature sensitivity, and their corresponding peptides, are providedherein. For example, host mesophilic bacteria can be transformed withone or more psychrophile TS essential nucleic acid molecules, therebyconferring TS to the mesophilic bacteria. The resulting recombinantmesophilic bacteria can be formulated into an immunogenic composition,to treat or prevent infection by the meosophilic bacteria. For example,recombinant mesophilic M. tuburculosis bacterium containing one or morepsychrophile TS essential nucleic acid molecules can be used to treat orprevent tuberculosis. The same approach can be used to make TS forms ofBacillus anthracis, Brucella abortus, Burkholderia pseudomallei,Haemophilus influenzae, Mycobacterium bovis, Salmonella typhi, Shigelladysenteriae, Staphylococcus aureus, Streptococcus pneumoniae, andYersinia pestis which cause anthrax, brucellosis, melioidosis,meningitis, bovine tuberculosis, typhoid fever, dysentery, numeroustypes of nosocomial infections, pneumonia, and plague. Thus, such TSbacteria can be used to treat or prevent such conditions.

Temperature-sensitive essential proteins from a psychrophilic bacteriumare provided herein, such as those from Colwellia sp., Psuedoalteromonassp., or Shewanella sp. Exemplarily proteins include ligA, pyrG, hemC,ftsZ, cmk, murG, fmt, and dnaK. Exemplary sequences are provided in theamino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, and 28. However, one skilled in the art will appreciatethat variant sequences can also be used. For example, a peptide having asequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to the amino acid sequence set forth in one of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 is encompassed bythe present disclosure, and can be used in the methods provided herein.Variant sequences retain the biological activity of the nativetemperature-sensitive essential protein from a psychrophilic bacterium,such as conferring the ability to make a bacterium TS (such as heatsensitivity), for example operable at a temperature of −10° C. to about30° C. (such as 0° C. to 30° C.), but inoperable at a temperaturegreater than about 30° C. (for example 4° C. to 30° C.), such as greaterthan 35° C. Exemplary sequences can be obtained using computer programsthat are readily available on the internet and the amino acid sequencesset forth herein. In one example, the variant peptide retains a functionof the native protein, such as the ability to confer temperaturesensitivity to a bacterium.

A specific, non-limiting example of a variant protein is a conservativevariant of the native protein (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, and 28). Substitutions of the amino acidssequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, and 28 can be made based on this table, as long as thepathogenic mesophilic bacteria are rendered TS and are able to initiatean immune response to its pathogenic antigens. For example, proteinsequences can be altered without significantly altering their biologicalproperties, for example by introducing one or more conservative aminoacid substitutions. Therefore, any of SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, or 28 can be modified by making 1 to 20, 1to 15, 1 to 12, 1 to 10, or 1 to 5 conservative amino acidsubstitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or 50 conservative amino acid substitutions,while retaining the ability to render a mesophilic bacteria temperaturesensitive (TS). Examples of conservative substitutions are shown below:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Minor modifications to the disclosed protein sequences can result inpeptides which have substantially equivalent activity as compared to theunmodified counterpart protein described herein. Such modifications maybe deliberate, as by site-directed mutagenesis, or may be spontaneous.All of the proteins produced by these modifications are included herein.

Temperature-sensitive essential proteins (and nucleic acid molecules)from a psychrophilic bacterium are disclosed herein that can be used toinduce temperature sensitivity in a desired bacterial host, wherein theresulting recombinant bacteria can be used to induce an immune response(for example in a mammal). These peptides can include fragments of thefull-length native protein, as long as the ability to confer temperaturesensitivity in the host cell is retained. In these examples, the peptidedoes not include the full-length amino acid sequences set forth as 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28. For example no morethan 10%, no more than 5%, or no more than 1% of the amino acids can bedeleted, such as 1% to 5% of the amino acids.

The isolated temperature-sensitive essential proteins can be part of afusion protein. Thus, the fusion protein can include thetemperature-sensitive essential protein (see above) and a secondheterologous moiety, such as a myc protein, an enzyme or a carrier (suchas a hepatitis carrier protein or bovine serum albumin) covalentlylinked to the temperature-sensitive essential protein. In additionalexamples, the temperature-sensitive essential protein includes sixsequential histidine residues, a f3-galactosidase amino acid sequence,or an immunoglobulin amino acid sequence, for example at the C- orN-terminus of the temperature-sensitive essential protein. Thetemperature-sensitive essential protein can also be covalently linked toa carrier. Suitable carriers include, but are not limited to, ahepatitis B small envelope protein HBsAg.

The temperature-sensitive essential proteins disclosed herein can bechemically synthesized by standard methods, or can be producedrecombinantly. An exemplary process for polypeptide production isdescribed in Lu et al., Federation of European Biochemical SocietiesLetters. 429:31-35, 1998. Proteins can also be produced using moleculargenetic techniques, such as by inserting a nucleic acid encoding atemperature-sensitive essential protein into an expression vector,introducing the expression vector into a host cell. They can also beisolated by methods including preparative chromatography andimmunological separations.

Temperature-sensitive essential nucleic acid molecules from apsychrophilic bacterium are provided herein. Exemplary sequences areprovided in the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, and 27. However, one skilled in the artwill appreciate that variant sequences can also be used. For example, anucleic acid molecule having a sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% identical to the nucleic acid sequenceset forth in one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25 and 27 (such as at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to the nucleic acid sequence set forth innucleotides 10-2067 of SEQ ID NO: 1, nucleotides 10-2019 of SEQ ID NO:3, nucleotides 10-2019 of SEQ ID NO: 5, nucleotides 10-2010 of SEQ IDNO: 7) is encompassed by the present disclosure, and can be used in themethods provided herein. In some examples, the codons of a nucleic acidmolecule are optimized for the bacterium into which it is introduced. Insome examples, such optimization does not alter the amino acid sequenceencoded thereby. For example, the psychrophilic bacterium TS essentialnucleic acid can be modified to optimize codon usage for the mesophilicbacterium (e.g., M. tuberculosis or F. novicida) into which thepsychrophilic bacterium TS essential nucleic acid is introduced.Exemplary sequences can be obtained using computer programs that arereadily available on the internet and the nucleic acid sequences setforth herein. In one example, the variant nucleic acid sequence retainsthe ability to encode a protein having the function of the nativeprotein, such as the ability to confer temperature sensitivity (e.g.,heat sensitivity) to a mesophilic bacterium.

The disclosed temperature-sensitive essential nucleic acid moleculesfrom a psychrophilic bacterium include DNA, cDNA and RNA sequences whichencode the temperature-sensitive essential peptide. Silent mutations inthe coding sequence result from the degeneracy (i.e., redundancy) of thegenetic code, whereby more than one codon can encode the same amino acidresidue. Thus, for example, leucine can be encoded by CTT, CTC, CTA,CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, orAGC; asparagine can be encoded by AAT or AAC; aspartic acid can beencoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alaninecan be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAAor CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can beencoded by ATT, ATC, or ATA. Tables showing the standard genetic codecan be found in various sources (e.g., L. Stryer, 1988, Biochemistry,3.sup.rd Edition, W.H. 5 Freeman and Co., NY).

A nucleic acid molecule encoding a temperature-sensitive essentialpeptide from a psychrophilic bacterium can be cloned or amplified by invitro methods, such as the polymerase chain reaction (PCR), the ligasechain reaction (LCR), the transcription-based amplification system(TAS), the self-sustained sequence replication system (3SR) and the Qβreplicase amplification system (QB). For example, a polynucleotideencoding the protein can be isolated by polymerase chain reaction ofcDNA using primers based on the DNA sequence of the molecule. A widevariety of cloning and in vitro amplification methodologies are wellknown to persons skilled in the art. PCR methods are described in, forexample, U.S. Pat. No. 4,683,195; Mullis et al., Cold Spring HarborSymp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCR Technology,(Stockton Press, NY, 1989). Polynucleotides also can be isolated byscreening genomic or cDNA libraries with probes selected from thesequences of the desired polynucleotide under stringent hybridizationconditions.

The nucleic acid molecules encoding a temperature-sensitive essentialpeptide from a psychrophilic bacterium include a recombinant DNA whichis incorporated into a vector, into an autonomously replicating plasmidor virus, or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (such as a cDNA) independent of othersequences. The nucleic acid molecules disclosed herein can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double forms of DNA.

The nucleic acid molecules encoding a temperature-sensitive essentialpeptide from a psychrophilic bacterium can be part of a vector, such asa plasmid or viral vector. Suitable vectors include retrovirus vectors,orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors,suipox vectors, adenoviral vectors, herpes virus vectors, alpha virusvectors, baculovirus vectors, Sindbis virus vectors, vaccinia virusvectors and poliovirus vectors. Specific exemplary vectors are poxvirusvectors such as vaccinia virus, fowlpox virus and a highly attenuatedvaccinia virus (MVA), adenovirus, baculovirus and the like. Other viralvectors that can be used include other DNA viruses such as herpes virusand adenoviruses, and RNA viruses such as retroviruses and polio.

The nucleic acid molecules encoding a temperature-sensitive essentialpeptide from a psychrophilic bacterium can be operably linked to atleast one expression control element. The expression control elementsare inserted in the vector or plasmid to control and regulate theexpression of the nucleic acid sequence. For example, an expressioncontrol sequence operatively linked to a temperature-sensitive essentialpeptide coding sequence is ligated such that expression of the codingsequence is achieved under conditions compatible with the expressioncontrol sequences. The expression control sequences include, but are notlimited to, appropriate promoters, enhancers, transcription terminators,a start codon (i.e., ATG) in front of a protein-encoding gene, splicingsignal for introns, maintenance of the correct reading frame of thatgene to permit proper translation of mRNA, and stop codons. Specificexamples of expression control elements include, but are not limited to,lac system, operator and promoter regions of phage lambda, yeastpromoters and promoters derived from polyoma, adenovirus, retrovirus orSV40. Additional operational elements include, but are not limited to,leader sequence, termination codons, polyadenylation signals and anyother sequences necessary for the appropriate transcription andsubsequent translation of the nucleic acid sequence encoding thetemperature-sensitive essential peptide from a psychrophilic bacteriumin the host system. The expression vector can contain additionalelements necessary for the transfer and subsequent replication of theexpression vector containing the nucleic acid sequence in the hostsystem. Examples of such elements include, but are not limited to,origins of replication and selectable markers. It will further beunderstood by one skilled in the art that such vectors are easilyconstructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology,” John Wiley and Sons, New York,N.Y.) and are commercially available.

In one example, vector introduced into a host bacterium includes one ormore of the following elements: (i) a prokaryotic origin of replication,so that the vector may be amplified in a prokaryotic host; (ii) a geneencoding a marker which allows selection of prokaryotic host cells thatcontain the vector (e.g., a gene encoding antibiotic resistance); (iii)at least one DNA sequence encoding one or more temperature-sensitiveessential peptides from a psychrophilic bacterium located adjacent to atranscriptional promoter capable of directing the expression of thesequence; and (iv) DNA sequences homologous to the region of the parentvirus genome where the foreign gene(s) will be inserted, flanking theconstruct of element (iii).

The vector can contain an additional gene that encodes a marker thatwill allow identification of recombinant cells containing insertedforeign DNA. These include genes that encode antibiotic or chemicalresistance (e.g., see Spyropoulos et al., 1988, J. Virol. 62:1046;Falkner and Moss, 1988, J. Virol. 62:1849; Franke et al., 1985, Mol.Cell. Biol. 5:1918), as well as genes such as the E. coli lacZ gene,that permits identification of recombinant plaques by colorimetricassay.

Methods of introducing nucleic acid molecules, such as those that encodea temperature-sensitive essential peptide from a psychrophilicbacterium, are well known to those skilled in the art. Where the host isprokaryotic, such as, a bacterium, competent cells which are capable ofDNA uptake can be prepared from cells harvested after exponential growthphase and subsequently treated by the CaCl₂ method using procedures wellknown in the art. Alternatively, MgCl₂ or RbC1 can be used.Transformation can also be performed after forming a protoplast of thehost cell if desired, or by electroporation. Hosts cells can includebacterial cells, such as bacteria that cause disease. Examples of suchbacteria that can be used as host cells for temperature-sensitiveessential nucleic acids/peptides from a psychrophilic bacterium includewithout limitation any one or more of (or any combination of)Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomycessp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonassp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria(Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum,Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillusactinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis,Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillusstearothermophilus), Bacteroides sp. (such as Bacteroides fragilis),Bartonella sp. (such as Bartonella bacilliformis and Bartonellahenselae, Bifidobacterium sp., Bordetella sp. (such as Bordetellapertussis, Bordetella parapertussis, and Bordetella bronchiseptica),Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi),Brucella sp. (such as Brucella abortus, Brucella canis, Brucellamelintensis and Brucella suis), Burkholderia sp. (such as Burkholderiapseudomallei and Burkholderia cepacia), Campylobacter sp. (such asCampylobacter jejuni, Campylobacter coli, Campylobacter lari andCampylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis,Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci,Citrobacter sp. Coxiella bumetii, Corynebacterium sp. (such as,Corynebacterium diphtheriae, Corynebacterium jeikeum andCorynebacterium), Clostridium sp. (such as Clostridium perfringens,Clostridium difficile, Clostridium botulinum and Clostridium tetani),Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes,Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli,including opportunistic Escherichia coli, such as enterotoxigenic E.coli, enteroinvasive E. coli, enteropathogenic E. coli,enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenicE. coli) Enterococcus sp. (such as Enterococcus faecalis andEnterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia andEhrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp.,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae,Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae,Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobactersp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacterfennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiellapneumoniae, Klebsiella granulomatis and Klebsiella oxytoca),Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans,Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp.,Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp.,Mycobacterium sp. (such as Mycobacterium leprae, Mycobacteriumtuberculosis, Mycobacterium intracellulare, Mycobacterium avium,Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp. (such asMycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium),Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica andNocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae andNeisseria meningitidis), Pasteurella multocida, Plesiomonasshigelloides. Prevotella sp., Porphyromonas sp., Prevotellamelaminogenica, Proteus sp. (such as Proteus vulgaris and Proteusmirabilis), Providencia sp. (such as Providencia alcalifaciens,Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa,Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such asRickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii,Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) andRickettsia typhi), Rhodococcus sp., Serratia marcescens,Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonellaenterica, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella cholerasuis and Salmonella typhimurium),Serratia sp. (such as Serratia marcesans and Serratia liquifaciens),Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigellaboydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcuspneumoniae (for example chloramphenicol-resistant serotype 4Streptococcus pneumoniae, spectinomycin-resistant serotype 6BStreptococcus pneumoniae, streptomycin-resistant serotype 9VStreptococcus pneumoniae, erythromycin-resistant serotype 14Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcuspneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such asTreponema carateum, Treponema petenue, Treponema pallidum and Treponemaendemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonellasp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibriovulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrioalginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibriometchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia sp. (such asYersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis) and Xanthomonas maltophilia among others.

Following transformation of bacterial cells, recombinant host cells canbe identified by one of several techniques. For example, expression of agene encoding a marker or indicator gene with the temperature-sensitivegene, as described above, can be used to identify recombinant progeny.One specific non-limiting example of an indicator gene is the E. colilacZ gene. Recombinant bacterial cells expressing beta-galactosidase canbe selected using a chromogenic substrate for the enzyme. Once arecombinant bacterium has been identified, it can be selected andamplified for use in an immunogenic composition provided herein.

Methods of Making Temperature-Sensitive Bacterial Strains

The exemplary embodiments relate to methods for generating recombinantTS bacteria for use in stimulating an immune response to the TSbacteria. In one aspect, an exemplary TS immunogenic composition issuitable for immunoprophylaxis to prevent infectious disease oralternatively immunotherapy to treat an infectious disease. Such TSbacteria are generated by the introduction of one or more TS essentialgenes from psychrophilic bacteria into a target bacteria (such as amesophilic bacteria that causes a disease that one wants to treat orprevent). Thus, the disclosure provides safe immunogenic compositionsbased on live genetically altered bacterial microorganisms. This wasaccomplished by taking advantage of essential genes from psychrophilicbacteria, by creating a fusion of the psychrophilic structural geneswith the transcriptional and translational control elements of the“host” genome or by making fusions between the host gene and thepsychrophilic gene. The exemplary embodiments provide live vaccines andimmunogenic compositions that mimic a number of cold adapted viralvaccines and are unable to grow at the normal body temperature.

According to another exemplary embodiment it is suitable for massproduction purposes, specifically of antigen; due to the TS strain'snon-virulent nature the aerosols produced are rendered harmless andtherefore, this methods and compositions disclosed herein cansignificantly reduce or eliminate human risk of infection.

Another aspect, the methods and compositions provided herein has valueas a research diagnostic, or as a research/educational tool because itallows for experimentation to be performed on organisms that arenormally highly pathogenic in their viable state without posing threatsto the researcher.

The methods and compositions provided herein can be employed tostimulate the immune system with TS organisms with the intention ofprevention or treatment of a disease.

A large number of psychrophilic bacteria contain TS genes, which can beused to generate TS mesophilic bacteria of the present disclosure. Forexample, one or more TS essential genes from psychrophilic bacteria canbe introduced into a mesophilic bacterium (for example into a chromosomeof a mesophilic bacteria), thereby generating a TS strain that can beused to induce an immune response in a subject into whom it isadministered. Recombinant methods for introducing a nucleic acid intobacteria are routine in the art. Appropriate TS essential genes frompsychrophilic bacteria can be identified using the methods providedherein. As shown in Tables 1 and 2, nine of the twenty one essentialgenes from the psychrophilic C. psychrerhraea were introduced into F.novicida and substituted for an essential host gene to generate TSstrains of F. novicida (“Group I”). Group I genes generated a range ofTS phenotypes with the restrictive temperatures of about 33° C. to 44°C. Thus, the genes of Group I can be used to generate TS strains of thepresent disclosure. Group II in Table 1 consists of the C. psychrerhraeagenes that either functioned poorly or not at all in the exemplarybacterial strain F. novicida. F. novicida strains carrying an integratewith the psychrophilic essential gene resolve the integrate undercounter selection pressure generated by the presence of sacB andsucrose. However, the resolved strains retain copies of both thepsychrophilic gene and the F. novicida homologue and the strains are notTS (“Group III” in Table 1); indicating that these psychrophilicessential genes do not function in the mesophilic host. Alleles of thesame gene from different psychrophilic bacteria can be selected toidentify those that generate hybrid strains with the same TS propertieswhen substituted into the chromosomes of mesophilic bacteria. The ligAalleles from three different psychrophilic bacteria generated threedifferent TS phenotypes when substituted into the mesophile F. novicida.The pyrG_(Cp) allele from C. psychrerhraea created a TS strain whensubstituted into F. novicida but the pyrG_(Sf) allele from S.frigidimarina (SF) did not. PH refers to P. haloplanktis.

TABLE 1 Restrictive Gene Temp.(° C.) symbol Source Product FunctionGroup ligA_(Ph2) 28/PH NAD-dependent DNA ligase I ligA_(Sf) 33/SFNAD-dependent DNA ligase I ligA_(Cp) 34/CP NAD-dependent DNA ligase IligA_(Ph) 36.8/PH NAD-dependent DNA ligase I hemC_(Cp) 36.8/CPPorphobilinogen deaminase I (Hydroxymethylbilane synthase) pyrG_(Cp)37.2/CP CTP synthetase I dnaK_(Cp) 38.2/CP Molecular chaperone DnaK ImurG_(Cp) 38.2/CP UDP-N-acetylglucosamine-N- Iacetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase fmt_(Cp) 41/CP Methionyl-tRNAformyltransferase I ftsZ_(Cp) 42/CP Cell division protein I cmk_(Cp)43/CP Cytidylate kinase I tyrS_(Cp) 44/CP Aminoacyl tRNA synthetases forTyr I adk_(Cp) >44/CP Adenylate kinase (proved resolution) IIaccD_(Cp) >44/CP AcetylCoA carboxylase. The F. novicida II integratecontaining accD_(Cp) fails to resolve. murI_(Cp) >44/CP Glutamateracemase. The CP version of II MurI appears to function poorly at alltemperatures in F. novicida. pyrG_(Sf) >44/SF CTP synthetase IIItrxA_(Cp) >44/CP Thioredoxin III g/mS_(Cp) >44/CPGlucosamine—fructose-6-phosphate III aminotransferase argS_(Cp) >44/CPAminoacyl tRNA synthetases for Arg III cds_(Cp) >44/CP phosphatidatecytidylyltransferase III mur_(Cp)C >44/CP UDP-N-acetylmuramate—alanineligase III valS_(Cp) >44/CP Aminoacyl tRNA synthetases for Val IIIproS_(Cp) >44/CP Aminoacyl tRNA synthetases for Pro III metK_(Cp) ≦44/CPS-adenosylmethionine synthetase III ftsW_(Cp) >44/CP Cell divisionprotein III

TABLE 2 Mutation rate in F. novicida to temperature resistance Re- Chal-stricted lenge Temp Temp Gene (° C.) (° C.) Trial #1 Trial #2 Trial #3ligA_(Sf) 33 37  4.0 × 10⁻⁶  3.3 × 10⁻⁷  9.7 × 10⁻⁷ ligA_(Cp) 34 37 <1.2× 10⁻¹⁰ <7.93 × 10⁻¹¹  <1.1 × 10⁻¹⁰ ligA_(Ph) 36.8 39 <1.5 × 10⁻¹⁰ <7.8× 10⁻¹¹ <6.2 × 10⁻¹¹ dnaK_(Cp) 38.2 39.5 <3.2 × 10⁻¹⁰ <1.9 × 10⁻¹⁰ <3.2× 10⁻¹⁰ hemC_(Cp) 36.8 43 <2.5 × 10⁻¹⁰ <3.6 × 10⁻¹¹ <3.7 × 10⁻¹¹pyrG_(Cp) 37.2 40  8.5 × 10⁻⁸  1.0 × 10⁻⁹  6.5 × 10⁻⁸ murG_(Cp) 38.2 43 2.6 × 10⁻⁴  3.0 × 10⁻⁵  8.5 × 10⁻⁵ dnaK_(Sf) 39 42  3.1 × 10⁻¹⁰  8.5 ×10⁻¹⁰

To make a TS bacterial pathogen, an essential gene from an Arcticpsychrophile bacterium was substituted into the genome of a mesophilicpathogenic bacterium. The Arctic bacterial essential gene ligA_(Sf)rendered F. novicida unable to grow at a temperature of 33° C. orhigher. Table 2 outlines the restrictive temperature properties imposedon F. novicida following the replacement of the mesophilic essentialgene for its psychrophilic counterpart. Any of the genes in Table 2 maybe introduced into a pathogenic bacteria strain to create liveheat-sensitive vaccines. Exemplary pathogenic bacteria include but arenot limited to: Mycobacterium sp., Haemophilus sp., Vibrio sp.,Escherichia sp., Salmonella sp., Streptococcus sp., Burkholderia sp.,Campylobacter sp., Neisseria sp., and Francisella sp.

The disclosure relates to genes derived from psychrophilic bacteria foruse in the development of heat-sensitive immunogenic compositions, andmethods of using these compositions to stimulate an immune response in asubject. In a specific example, the disclosure provides recombinantpathogens (such as Mycobacterium sp., Haemophilus sp., Vibrio sp.,Escherichia sp., Salmonella sp., Streptococcus sp., Burkholderia sp.,Campylobacter sp., Neisseria sp., and Francisella sp.) containing one ormore heat-sensitive genes, exemplified by ligA, pyrG, hemC, ftsZ, cmk,dnaK, and fmt, that can be administered to a subject to provide aprophylactic immune response against diseases caused by such bacteria.

Methods of making a recombinant temperature-sensitive (TS) bacterialcell are provided. In one example the method includes introducing intothe genome of a mesophilic bacterial strain a nucleic acid constructthat includes a TS essential nucleic acid molecule from a psychrophilicbacteria (such as one that encodes a peptide that is operable at atemperature of about −10° C. to about 30° C., and/or inoperable at atemperature greater than about 30° C., for example Colwellia sp.,Psuedoalteromonas sp., or Shewanella sp) and one or more controlsequences operably linked to the TS essential nucleic acid molecule. Thetemperature-sensitive essential polynucleotide renders the mesophilicbacteria operable at a temperature less than about 30° C. and inoperableat a temperature greater than about 30° C. In some examples, thetemperature-sensitive essential nucleic acid molecule includes anucleotide sequence having at least 80%, at least 90%, or at least 95%sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. In some examples themethod also includes isolating the TS essential nucleic acid moleculefrom the genome of the psychrophilic bacterial strain. The method canalso include constructing or generating the nucleic acid constructcomprising the TS essential nucleic acid molecule and one or morecontrol sequences operably linked to the TS essential nucleic acidmolecule.

In some examples, the method further includes culturing the recombinantTS bacterial host cell at a temperature wherein thetemperature-sensitive peptide is operable, whereby said recombinant TSbacterial host cell produces a plurality of peptides; increasing theculturing temperature to a temperature at which thetemperature-sensitive peptide is inoperable; maintaining said culturingfor a period of time sufficient to kill the recombinant TS bacterialhost cell; and harvesting the killed recombinant TS bacterial hostcells.

Methods of making a recombinant TS bacterial host cell can also includethe following. A psychrophilic microbial genome is screened fordetection of a TS essential polynucleotide that encodes a peptide thatis inactivated at about greater than 30° C.; isolating said TS essentialpolynucleotide; constructing a nucleic acid construct comprising the TSessential polynucleotide and one or more control sequences operablylinked to the TS polynucleotide; inserting the nucleic acid constructinto the genome of a selected mesophilic bacterial host cell (such asFrancisella novicida) thereby functionally replacing the host cell'shomologue of the TS essential polynucleotide whereby the TS peptide (andthus the bacteria in which it is expressed) is operable at a temperatureless than about 30° C., and inoperable at a temperature greater thanabout 30° C. and mimics the temperature sensitivity of the originaldesignated host bacterium. The resulting recombinant mesophilicbacterial host cell comprising the TS polynucleotide is cultured orgrown at a temperature less than about 30° C. to confirm the viabilityof the recombinant mesophilic bacterial host cell; further culturing therecombinant mesophilic bacterial host cell comprising the TSpolynucleotide at a temperature greater than about 30° C. to determineif the mesophilic bacterial host cell is killed. If the mesophilicbacterial host cell is killed, the nucleic acid construct is introducedinto the genome of a selected destination mesophilic bacterial host cell(such as Salmonella sp. or Mycobacterium sp.) thereby functionallyreplacing the host cell's homologue of the temperature-sensitiveessential polynucleotide whereby the temperature-sensitive peptide (andthus the bacteria in which it is expressed) is operable at a temperatureless than about 30° C., and inoperable at a temperature greater thanabout 30° C. and mimics the temperature sensitivity of the originaltester host bacterium.

In some examples, the mesophilic bacteria is one that is operable at atemperature selected from the range of about 10° C. to about 50° C.prior to introduction of the TS essential nucleic acid molecule from apsychrophilic bacteria. Examples of such mesophilic bacteria includestrains of fermentative bacteria or bioremediation bacteria. Otherexemplary bacteria are provided above.

In some examples, the TS essential nucleic acid molecule expresses apeptide during a culturing of the recombinant TS bacteria, such as apeptide having at least 80%, at least 90%, or at least 95% sequenceidentity to an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, or 28.

Recombinant TS bacteria generated by these methods, as well ascompositions that include such bacteria, are also provided herein.

Temperature-Sensitive Bacterial Strain Compositions

Compositions are provided that include recombinant TS bacteria providedherein. In some examples, the compositions include more than one type ofrecombinant TS bacteria, such as 2, 3, 4 or 5 different recombinant TSbacteria. In some examples, the recombinant TS bacteria contain two ormore different TS essential psychrophilic coding sequences (such as twoor more of the Group I genes listed in Table 1, such as ligA and anotherGroup I gene). In particular examples, the recombinant TS bacteria is aFrancisella sp., Salmonella sp., or Mycobacterium sp. (other particularexamples are provided above).

In some examples, such compositions are immunogenic, in that they canstimulate an immune response in a mammal. The compositions can includeother components, such as pharmaceutically acceptable carriers (such assaline), adjuvants, preservatives, combinations thereof, and the like.

Methods of Stimulating an Immune Response Using Temperature-SensitiveBacterial Strains

The TS recombinant bacteria disclosed herein can be used to generate animmune response in a subject. In some examples, the subject is infectedwith a bacterium, or as at risk of being infected with a bacterium (suchas a health care worker), such as Mycobacterium tuberculosis. Thus, inseveral embodiments, the methods include administering to a subject atherapeutically effective amount of one or more of the TS recombinantbacteria disclosed herein in order to generate an immune response, suchas, but not limited to, a protective immune response. For example, twoor more different TS recombinant bacteria (such as those expressingdifferent TS essential peptides from psychrophilic bacteria) can be usedto generate an immune response in a subject. In some examples, therecombinant bacterium used to generate an immune response in a subjectexpresses two or more different temperature-sensitive essential peptidesfrom a psychrophilic bacterium or the same temperature-sensitiveessential peptide from two or more different psychrophilic bacteria.

The TS recombinant bacterium administered is selected based on thebacterial infection to be prevented or treated. For example, if thebacterial infection to be prevented or treated in the subject istuberculosis, then the TS recombinant bacteria is Mycobacteriumtuberculosis expressing at least one TS essential peptide from apsychrophilic bacterium. In another example, if the bacterial infectionto be prevented or treated in the subject is tularemia, then the TSrecombinant bacteria is F. tularensis expressing at least one TSessential peptide from a psychrophilic bacterium.

In exemplary applications, compositions are administered to a subjecthaving in an amount sufficient to produce an immune response to the TSrecombinant bacteria. These TS recombinant bacteria are of use toprevent a bacterial infection (such as Mycobacterium tuberculosis)prevent progression to disease in a subject having a latent bacterialinfection, or to treat a disease resulting from the bacterial infection(such as tuberculosis). In several examples, administration of atherapeutically effective amount of a composition including the TSrecombinant bacteria disclosed herein induces a sufficient immuneresponse to decrease a symptom of a disease due to bacterial infection,to prevent the development of one or more symptoms of the diseaseassociated with the infection, or to prevent infection with thebacteria.

In some examples, the compositions are of use in preventing a futurebacterial infection. Thus, a therapeutically effective amount of thecomposition is administered to a subject at risk of becoming infectedwith a bacterium, such as Mycobacterium tuberculosis. For example thedisclosed compositions can be used to prevent the development oftuberculosis, such as latent or active tuberculosis in the subject uponsubsequent exposure to Mycobacterium tuberculosis. In one example, thecompositions are administered to a subject with a latent Mycobacteriumtuberculosis infection, and prevent the development of symptoms oftuberculosis. Thus the compositions are of use in treating a subjectwith latent tuberculosis, such that the subject does not develop activetuberculosis.

Amounts effective for these uses will depend upon the severity of thedisease, the general state of the patient's health, and the robustnessof the patient's immune system. In one example, a therapeuticallyeffective amount of the compound is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Inother examples, a therapeutically effective amount is an amountsufficient to prevent an infection with the bacterium in a subject uponsubsequent exposure of the subject to the bacterium. In additionalexamples, a therapeutically effective amount is an amount sufficient toprevent development of symptom in a subject infected with a bacterium.

The TS recombinant bacteria-containing composition can be administeredby any means known to one of skill in the art either locally orsystemically, such as by intramuscular injection, subcutaneousinjection, intraperitoneal infection, intravenous injection, oraladministration, nasal administration, transdermal administration or evenanal administration. In one embodiment, administration is by oral,subcutaneous injection or intramuscular injection. To extend the timeduring which the TS recombinant bacteria is available to stimulate aresponse, the TS recombinant bacteria can be provided as an implant, anoily injection, or as a particulate system. The particulate system canbe a microparticle, a microcapsule, a microsphere, a nanocapsule, orsimilar particle. A particulate carrier based on a synthetic polymer hasbeen shown to act as an adjuvant to enhance the immune response, inaddition to providing a controlled release. Aluminum salts can also beused as adjuvants to produce an immune response.

In one specific, non-limiting example, the TS recombinant bacteria areadministered in a manner to direct the immune response to a cellularresponse (that is, a cytotoxic T lymphocyte (CTL) response), rather thana humoral (antibody) response.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72,B7-1, B7-2, or other B7 related molecules; one or more molecules such asOX-40L or 41 BBL, or combinations of these molecules, can be used asbiological adjuvants (see, for example, Salgaller et al., 1998, J. Surg.Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J. Sci. Am. 6(Suppl1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper etal., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can beadministered systemically (or locally) to the subject. In some examples,IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1B7-2, OX-40L, 41 BBL and ICAM-1 are administered. In variousembodiments, the nucleic acid encoding the biological adjuvant can becloned into same vector as the psychrophilic TS essential peptide codingsequence, or the nucleic acid can be cloned into one or more separatevectors for co-administration into the bacteria.

A pharmaceutical composition including TS recombinant bacteria is thusprovided. These compositions are of use to promote an immune response toa particular bacterium. In one embodiment, TS recombinant bacteria aremixed with an adjuvant containing two or more of a stabilizingdetergent, a micelle-forming agent, and an oil. Suitable stabilizingdetergents, micelle-forming agents, and oils are detailed in U.S. Pat.Nos. 5,585,103; 5,709,860; 5,270,202; and 5,695,770, all of which areincorporated by reference. A stabilizing detergent is any detergent thatallows the components of the emulsion to remain as a stable emulsion.Such detergents include polysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,ZWITTERGENT™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by BASF Wyandotte publications, e.g., Schmolka, J.Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al., J. Immunol.129:1244, 1981, PLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of suchagents are well known in the art. In one embodiment, the agent is chosento have a hydrophile-lipophile balance (HLB) of between 0 and 2, asdefined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent canbe provided in an effective amount, for example between 0.5 and 10%, orin an amount between 1.25 and 5%.

In one example oil is included in the composition. Examples of such oilsinclude squalene, Squalane, EICOSANE™, tetratetracontane, glycerol, andpeanut oil or other vegetable oils. In one specific, non-limitingexample, the oil is provided in an amount between 1 and 10%, or between2.5 and 5%. The oil should be both biodegradable and biocompatible sothat the body can break down the oil over time, and so that no adverseaffects, such as granulomas, are evident upon use of the oil.

In one embodiment, the adjuvant in the composition is a mixture ofstabilizing detergents, micelle-forming agent, and oil available underthe name PROVAX® (IDEC Pharmaceuticals, San Diego, Calif.). An adjuvantcan also be an immunostimulatory nucleic acid, such as a nucleic acidincluding a CpG motif, or a biological adjuvant (see above).

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems, see Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., 1995. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres, the therapeutic agent is dispersedthroughout the particle. Particles, microspheres, and microcapsulessmaller than about 1 μm are generally referred to as nanoparticles,nanospheres, and nanocapsules, respectively. Capillaries have a diameterof approximately 5 μm so that only nanoparticles are administeredintravenously. Microparticles are typically around 100 μm in diameterand are administered subcutaneously or intramuscularly (see Kreuter,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

In particular examples, at least 10² CFU of the TS bacteria disclosedherein are administered per dose, such as at least 10³ CFU, at least 10⁴CFU, at least 10⁵ CFU, at least 10⁶ CFU, at least 10⁷ CFU, at least 10⁸CFU, such as10² to 10⁸ CFU or 10⁴ to 10⁸ CFU. In particular examples,such dosages are administered intradermal or intranasal.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until atherapeutic result is achieved. In one embodiment, the dose issufficient to treat or ameliorate symptoms or signs of bacterialinfection without producing unacceptable toxicity to the subject. Inanother embodiment, the dose is sufficient to prevent infection with abacterium upon subsequent exposure to the bacterium (such as M.tuberculosis). In a further embodiment, the dose is sufficient toprevent a symptom of bacterial infection (e.g., tuberculosis) in asubject with a latent bacterial infection. Systemic or localadministration can be utilized.

Thus the disclosure provides methods for producing an immune response toa bacterium in a subject. The method can include administering to thesubject a therapeutically effective amount of a TS bacterium, whereinthe temperature-sensitive bacterium expresses a psychrophilic TSessential protein or nucleic acid molecule provided herein (such as anucleic acid coding sequence in a vector), thereby inducing an immuneresponse to the bacterium. The method can further include administeringother agents, such as an adjuvant or antimicrobial agent (such as anantibiotic). In some examples, the immune response is a protectiveimmune response. The subject may have a bacterial infection, be at riskfor acquiring a bacterial infection, or have a latent bacterialinfection. Exemplary bacterial infections include infections with is M.tuberculosis, Salmonella or Francisella.

Methods of measuring an immune response following stimulation with abacterial antigen, such as a cytokine response, are known in the art. Insome examples, the method further includes measuring an immune responsefollowing administration of the therapeutic compositions providedherein. In one example, a cytokine response is increased followingadministration of the composition provided herein, such as an increaserelative to the absence of administration of the composition. In oneexample, cytokine production increases by at least 20%, such as at least40%, at least 50%, at least 75%, at least 90%, or at least 95% followingadministration of the composition, relative to the cytokine response inthe absence of administration of the composition.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLE 1

This example pertains to an exemplary method to create recombinantpsychrophilic genes joined to flanking DNA of a mesophilic host.

FIG. 1a exemplifies the fusion PCR (also known as, “extension overlapPCR”, “overlap PCR” or “splice overlap PCR”) strategy used toincorporate the C. psychrerythraea essential gene (C2) into the wt F.novicida genome. The C. psychrerythraea genes were engineered withoverlap PCR to contain the ribosome binding site (RBS) and the initialthree codons and stop codon of the surrounding F. novicida genes (F1 andF3) to promote translation of the C2 gene at normal levels for F.novicida. The fusion PCR product was ligated to an erythromycinresistant sacB cassette (Em^(R)-sacB) prior to its transformation intoF. novicida. Em^(R) colonies containing the fusion PCR product weregrown in the presence of sucrose and colonies were screened for the lossof Em^(R), the F. novicida essential gene (F2), and the presence of C2.

FIG. 1b illustrates the introduction of the psychrophilic gene fusionconstruct into the target organism's chromosome via a single crossoverevent. Furthermore, it illustrates that the excision can be enhancedusing the counter-selectable sacB marker. For genes that are not a partof a multi-cistronic operon the upstream pathogen genomic region wasfused to the psychrophilic structural gene from codon 4 through to thestop codon. A similar approach was used when substituting apsychrophilic allele into the middle of an operon. However, as oneskilled in the art can appreciate, depending on the nature of theoperon, some of the codons at the C terminus of the host homologueremained if they were important for translation of the downstreamcistron.

EXAMPLE 2

This example pertains to an exemplary method to insert the psychrophilicallele into the mesophilic bacteria.

FIG. 2a illustrates the substitution region for the psychrophilic ligAgene, corresponding to SEQ ID NO: 1. Additionally, it illustrates itsincorporation into the wt F. novicida chromosome. FIGS. 2b-e illustratethe integration point for the psychrophilic ligA genes of C.psychrerythraea, S. frigidmarina, P. haloplanktis I, and P. haloplanktis2 respectively. The first three codons for F. novicida were retained inorder to maximize the potential for ligA expression levels. FIG. 2a-eillustrates that in most cases the integration and excision eventsresult in a simple substitution of the psychrophilic gene for themesophilic host homologue. However, the integration and excision eventsmay also lead to the formation of a hybrid gene as illustrated in FIG. 1b.

EXAMPLE 3

This example pertains to an exemplary method to determine the maximalgrowth temperature of each bacterial strain and to show its growthproperties at restrictive temperatures.

Each bacterial strain was tested on agar plates placed in a highlystable (±1° C.) incubator; the restrictive temperatures were defined asthe lowest temperatures that did not permit the formation of isolatedcolonies on an agar streak plate. The growth properties at differenttemperatures of four different transgenic strains of F. novicidacarrying psychrophilic ligA substitutions and the growth properties ofwt F. novicida are shown in FIGS. 3-6. The psychrophilic ligA genes areligA_(Cp), ligA_(Sf), ligA_(Ph) and ligA_(Ph2), as represented by SEQ IDNOs: 1, 7, 3, and 5 respectively. In the first panels in FIGS. 3-6,growth is shown at a permissive temperature, i.e., a temperature belowthe restrictive temperature. In subsequent panels, growth of both thetransgenic and wt strains is shown before and after a shift to therestrictive temperature or higher.

Extended growth curves of both the F. novicida transgenic and wt strainsare shown as inserts in select panels of FIG. 3-6. These curves weregenerated by taking a fully grown culture, diluting it, and monitoringits growth in fresh growth media. More specifically, the wt and TStransgenic F. novicida cultures were grown at restrictive temperaturesuntil they reached stationary phase at which point, they were dilutedand re-incubated for growth at the restrictive temperatures again. Theadditional growth curves demonstrate that the cessation of growthexhibited by the transgenic strain is a real phenomenon, as opposed to atemporary adjustment to the temperature shift.

EXAMPLE 4

This pertains to an exemplary method used to determine the frequency ofmutations that permit bacterial growth at temperatures higher than therestrictive temperature of TS F. novicida transgenic strains.

Cultures were grown to late logarithmic phase at the permissive growthtemperature, they were then diluted in a series of 10⁹-10⁵ cells/plateon agar and incubated at temperatures about 3° C. above the restrictivetemperature, as well as at temperatures about 3° C. below therestrictive temperature. From this dilution series the rate of mutationsthat allow for growth at higher temperatures were calculated, Table 2exemplifies the frequency of mutation to temperature resistance in F.novicida. Remarkably, some of the psychrophilic genes are unable tomutate to forms that will function above their restrictive temperature.One skilled in the art may hypothesize that the millions of yearsrequired to adapt to a cold climate renders some of the psychrophilicessential gene products unable to adopt simple changes allowing them tofunction in temperatures typical to their mesophilic counterparts. Theseinclude ligACp, ligA_(Ph), hemC_(Cp), dnaK_(Cp), fmt_(Cp), anddnaK_(Sf).

EXAMPLE 5

This example pertains to an exemplary method to determine the durationof viability of the recombinant TS bacterial strains at the restrictivetemperature.

An exemplary culture of a TS transgenic strain that has a maximal growthtemperature of about 33° C., was grown at about 30° C. and a sample ofthe culture was incubated at about 37° C. to mimic the typicaltemperature of human body core tissues. Samples were taken at varyingtime points between 0-24 hours, and the individual samples werere-diluted, plated on to growth media, and then cultured at about 30° C.to determine the death rate above the restrictive temperature. As acontrol, the same experiment was carried out with the wt bacterium.

The persistence of F. tularensis strains carrying the psychrophilicessential genes within their macrophages was determined. Transgenicstrains were cultured at about 30° C. and used to infect macrophages atabout 37° C. in 24 well tissue culture plates using standard methodsknown to those skilled in these arts. For several days monitoring theinfected macrophages a subset of cells were lysed and the bacteria wereplated onto agar medium and incubated at about 30° C. The data generatedin these experiments showed the lifespan of transgene strains during aninfection with macrophages at a restrictive temperature and helped topredict the persistence of TS strains during infections.

This example can be extrapolated to provide an in vitro correlation forwhat can occur in a mammal. A TS transgenic strain will grow in a coolpart of the body such as the skin. Replication of the strain at andabout this cool site will constantly cause the TS transgenic strainprogeny to be moved into the draining lymph nodes. Depending on thelocations of the lymph nodes and the restrictive temperature of the TStransgenic strain, the TS progeny will die over a period of severalhours. The presence of the TS transgenic strain both in its live anddead states will stimulate an immune response.

EXAMPLE 6

This pertains to an exemplary method to determine the ability of a TSessential gene from a psychrophile to impart its TS phenotype on amesophilic bacterium. Specifically, it provides a method fortransferring a psychrophilic essential gene encoding a TS product to avariety of bacteria as well as the transfer of the TS essential genebetween mesophiles.

Several psychrophilic essential genes were substituted into the genomeof the mesophilic bacterium F. novicida. Multiple approaches can be usedto inserting a psychrophilic essential gene into a given bacterium inplace of its mesophilic homologue. Furthermore, it can be appreciatedthat one can substitute a given psychrophilic essential gene into manydifferent bacteria. The following three methods exemplify various waysof substituting ligA_(Cp) into three different bacteria. A commonapproach to gene substitution is illustrated in FIG. 1b , and involvesthe integration of a foreign gene in a bacterium that is in closeproximity to the hosts' homologous gene through PCR. Followingintegration, a counter selective marker, such as sacB, can be used tohelp identify the results of the integration and excision events.Specifically this approach was used to replace the F. novicida ligA genewith the psychrophilic ligA_(Cp) gene.

An alternate approach was used to replace the S. enterica ligA. Thestrain of S. enterica used had a bacteriophage Mu insertion in thechromosomal copy of ligA (Park et al., 1989. J. Bacteriol. 171:2173-80). A wt copy of the bacteriophage T4 DNA ligase was carried onthe ampicillin resistant plasmid, pBR313. The ligA_(Cp) gene wasintroduced on the compatible chloramphenicol resistant plasmid,pSUP2716, and the recombinant S. enterica strain was cultured in theabsence of ampicillin and the presence of chloramphenicol. These growthconditions allow the pBR313:T4 DNA ligase recombinant plasmid to belost. S. enterica strains that had lost the plasmid encoding the T4 DNAligase, rendering them ampicillin sensitive, were dependant on theligA_(Cp) for viability and were TS.

Another alternate approach can be employed when introducing apsychrophilic essential gene into Gram-positive bacteria. The method ofinsertion of ligA_(Cp) into M. smegmatis described herein exemplifiesthis method. A version of ligA_(Cp) (SEQ ID NOS: 17 and 18) designedwith optimal codons was cloned into the mycobacterial plasmid, pSM1;this a precautionary step due to the low G+C content in the ligA_(Cp)gene when compared to that of the M. smegmatis and M. tuberculosis ligAgenes. The recombinant pSMT3:ligA_(Cp) was electroporated into M.smegmatis. Subsequently a large fragment of the M. smegmatis ligA genewas deleted resulting in a strain dependent on ligA_(Cp) for viability.This strain was TS at about 34° C. This temperature is reflective of theTS nature of the F. novicida transgene strain encoding ligA_(Cp).

This example illustrates the use of a mesophilic tester strain whichcontains a psychrophilic essential gene to predict the TS phenotype whensaid psychrophilic essential gene is used to construct a transgenestrain of another mesophilic bacterium. In this example, the testerstrain was F. novicida. The substitution of ligA_(Cp) for the F.novicida ligA homologue showed that ligA_(Cp) functioned in themesophile and imparted a TS phenotype having a restrictive temperatureof about 34° C. The phenotype of the transgenic strain of F. novicidacarrying ligA_(Cp) predicted that substitution of the ligA_(Cp) geneinto other mesophiles (destination hosts) would results in viablebacteria that had a restrictive temperature of 34° C. The phenotype ofthe Salmonella and Mycobacteria transgene strains carrying ligA_(Cp)showed that the inter-genus transfer of a TS psychrophilic essentialgene could result in a phenotype seen in the tester strain.

EXAMPLE 7

This example describes an exemplary method to combine psychrophilicgenes or fragments thereof (as represented by SEQ ID NO 1-24) or mutantessential psychrophilic genes to create gene products with desired TSproperties.

Combining about 30%, at the 5′-end, of the novicida pyrG gene with about⅔ of the 3′-end of the C. psychrerythraea pyrG gene (pyrG_(Cp)) in theregion of codon 157-159 created a recombinant gene that was TS at 37° C.The F. novicida and C. psychrerythraea pyrG genes are identical atcodons 157-159 inclusive. Additionally, the single point mutation atamino acid residue 149 in ligA_(Ph) from an asparagine (“N”) residue toa lysine (“K”) residue changes the restrictive temperature from 37° C.to 28° C.

This approach could be applied to different psychrophilic genes by usingeither in vitro or in vivo recombinant technologies to combine two ormore homologues of the same gene.

EXAMPLE 8

This example pertains to an exemplary method to determine thedistribution of a transgenic strain from a site of infection in amammal.

F. novicida (a.k.a. F. tularensis subspecies novicida) carrying apsychrophilic transgene was used. One skilled in the art will appreciatethat similar methods can be used to generate and examine TS strains ofF. tularensis. F. novicida is highly virulent in mice. The infection ofmice by F. novicida serves as a model for the infection of largermammals with F. tularensis. Most strains of F. tularensis are highlyvirulent in most mammals.

The distribution of F. novicida transgenic strains from the site ofinfection was assessed either by injecting the recombinant strainsthrough the skin, or by introduction via the nose, and measuring theamount of viable F. novicida cells in internal organs such as the lung,liver and spleen about three to ten days after the inoculation. It wasfound that TS F. novicida transgenic strains did not spreadsignificantly from the site of inoculation. A direct correlation betweenthe inactivation temperature of the psychrophilic essential gene and thelevel of distribution throughout the system was observed; thedissemination of TS F. novicida strains is Lewis Rats is outlined inTable 3.

TABLE 3 Restrictive Temp. CFU/Tail F. novicida strain (° C.) injectionsite CFU/Spleen wt⁻ 45 9.7 × 10³/7.1 × 10³ 3.7 × 10⁶/2.2 × 10⁶ ligA_(Cp)34 5 × 10²/3 × 10² 0/0 ligA_(Ph) 36.8 3 × 10²/2 × 10² 0/0 dnaK_(Cp) 38.21.5 × 10⁴/7.6 × 10⁴ 5.0 × 10²/0     fmt_(Cp) 41 5.2 × 10³/2.4 × 10³ 3.5× 10⁵/2.1 × 10⁵

As a further example, one of the psychrophilic essential genes(ligA_(Cp)) was substituted into the genome of M. tuberculosis to createa transgenic strain. Some psychrophilic essential genes originate inbacteria with DNA with low G+C content. Thus the genes were optimizedwith codons for M. tuberculosis prior to inserting the psychrophilicgenes into the pathogenic bacteria (SEQ ID NOS: 17 and 18 provide theoptimized sequences). Codon optimization is a method well known to thoseskilled in these arts and can be accomplished using freely availablebioinformatic tools. The codon optimized psychrophilic essential geneswere inserted into M. tuberculosis by methods that are well described inExamples 1 and 2. M. tuberculosis, like M. smegmatis, are Gram-positivebacteria.

Another exemplary method pertains to an exemplary method thedistribution of a Gram-negative pathogenic strain. A psychrophilicessential gene was introduced into S. enterica. Upon introduction of theligA_(Cp) psycrophilic essential gene into S. enterica, the result was atransgenic strain that was unable to grow at 37° C., as illustrated inFIG. 8. Furthermore, this strain was unable to disperse from the site ofinoculation in infected mice, as evidenced by the inability of thestrain to migrate to the lungs, liver or spleen.

EXAMPLE 9

This example pertains to an exemplary method to determine the level ofprotective immune response generated from the inoculation of a mammalwith a TS transgenic bacterial strain. Methods of inoculation are knownin the art, and can include i.v., i.m., s.c., or i.p injection, as wellas inhalation, oral, and transdermal routes of delivery. One skilled inthe art will appreciate that methods similar to those described in thisexample can be used to test any transgenic TS bacterial strain thatincludes one or more psychrophilic essential nucleic acid sequences.

Inoculation of mice with a TS F. novicida transgenic strain(Fn-ligA_(Ph),Fn-ligA_(Cp) or Fn-dnaK_(Cp)) caused the cells of theirimmune systems to be stimulated (as measured by reduced bacterial organburdens) resulting in protection against infection with wt F. novicida(FIGS. 10a-d ). Mice were initially inoculated with the TS transgenicstrain and then challenged with an inoculation three weeks later of thewt F. novicida strain. This resulted in reduced growth in the livers andspleens of mice infected with the wt strains as compared to mice thathad not been inoculated with recombinant F. novicida. Furthermore,decreases in the morbidity and mortalities were observed among theinoculated group of mice resulting in the conclusion that immuneprotection was achieved.

Similarly, mice vaccinated with M. tuberculosis and S. entericatransgeneic strains (ligAPh) were shown to be more resistant toinfections with the wt pathogens than were un-vaccinated mice.

EXAMPLE 10

This example pertains to an exemplary method of discovering novelpsychrophilic essential genes.

Psychrophilic bacterium can be isolated from a cold environment, forexample ocean waters near the Earth's poles. Essential genes can beidentified by using degenerate PCR or other standard techniques to findhighly conserved genes, such as bacterial essential genes. Once thesegenes have been identified, they can be substituted into the genome of amesophile using the methods provided herein or known in the art,displacing the host homologue of the gene. The resulting strain can thenbe tested for temperature sensitivity as described herein.

EXAMPLE 11

This example pertains to an exemplary method of using TS transgenestrains in drug discovery research. Although a TS F. tularensis strainis exemplified, one skilled in the art will appreciate that similarmethods can be used for other TS strains generated using the methodsprovided herein.

A TS transgenic strain of F. tularensis (ligA_(Ph)) that was inoperableabove about 37° C. was used to infect cell line macrophages grown inmicrotiter plates at 34° C. A library of antimicrobial drug candidateswas introduced to individual wells that contained the infectedmacrophages, and the effect of the drug candidates on the killing of F.tularensis was measured by lysing the macrophages at various time pointsand determining the number of viable TS transgenic F. tularensis byplating on agar plates. Wt F. tularensis is extremely infectious andcauses a deadly disease. The use of the TS transgenic F. tularensisstrain allowed one to use greatly relaxed biological containmentconditions because the strain is incapable of causing disease in humans.

EXAMPLE 12

This example pertains to an exemplary method of generating and using TSstrains of Mycobacterium containing temperature-sensitive essentialnucleic acid molecules from psychrophilic bacteria to develop animmunogenic composition, which for example can be used to stimulate animmune response in a mammal, to protect or treat an M. tuberculosisinfection in the mammal.

The ligA_(Ph) and pryG_(Cp) genes will separately be introduced into M.tuberculosis H37Rv using an integration/excision approach. Thecounter-selectable marker sacB will be used to enhance the generation ofexcision events that can be detected. C57BL/6 mice will be vaccinated byintroducing 10,000 bacteria subcutaneously at the base of the tail.Negative controls mice injected with PBS and positive control miceinjected with the BCG strain will processed at the same time. The micewill be rested for 30 days. Following this period all of the mice willbe exposed to an aerosol of M. tuberculosis H37Rv that deposits 150bacteria into the lungs. At weeks 0, 4, 8, 16 and 32 following exposureto M. tuberculosis H37Rv, the mice will be euthanized and the number ofM. tuberculosis H37Rv in the lungs and spleens determined. If thetransgenes TS M. tuberculosis strains are successful at inducing aprotective immune response, the number of bacteria in the mice organswill be less than that of the negative control. Subsequent experimentswill be performed in a guinea pig model of tuberculosis.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that illustratedembodiments are only examples of the invention and should not beconsidered a limitation on the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

What is claimed is:
 1. A method of making a recombinant mesophilicbacterium that is temperature-sensitive (TS), comprising: introducinginto the genome of a mesophilic bacterium by homologous recombination anucleic acid construct comprising a TS essential nucleic acid moleculefrom a psychrophilic bacterium flanked on both sides by a nucleic acidmolecule homologous to a region of the mesophilic bacterium genome wherethe TS essential nucleic acid molecule from the psychrophilic bacteriumwill be inserted into the mesophilic bacterium genome, and functionallyreplacing the mesophilic bacterium's homolog of the TS essential nucleicacid molecule, thereby making a recombinant mesophilic bacterium that isTS, wherein a protein encoded by the TS essential nucleic acid moleculeis operable at a temperature less than 30° C. and inoperable at atemperature greater than 30° C., and wherein the recombinant mesophilicbacterium that is TS has a restrictive temperature between 33° C. and44° C.
 2. A method of making a killed recombinant mesophilic bacteriumthat is temperature-sensitive (TS), comprising: introducing into thegenome of a mesophilic bacterium a nucleic acid construct comprising aTS essential nucleic acid molecule from a psychrophilic bacteriumflanked on both sides by a nucleic acid molecule homologous to a regionof the mesophilic bacterium genome where the TS essential nucleic acidmolecule from the psychrophilic bacterium will be inserted into themesophilic bacterium genome, wherein a protein encoded by the TSessential nucleic acid molecule is operable at a temperature less than30° C. and inoperable at a temperature greater than 30° C., and whereinthe recombinant mesophilic bacterium that is TS is viable at atemperature of 0° C. to 30° C., nonviable at a temperature greater than30° C., and has a restrictive temperature between 33° C. and 44° C.;culturing the recombinant mesophilic bacterium that is TS at atemperature wherein a protein encoded by the TS essential nucleic acidmolecule is operable, whereby the recombinant mesophilic bacterium thatis TS produces a plurality of peptides; increasing the culturingtemperature to a temperature at which a protein encoded by the TSnucleic acid molecule is inoperable; maintaining said culturing for aperiod of time sufficient to kill the recombinant mesophilic bacteriumthat is TS; and harvesting the killed recombinant mesophilic bacteriumthat is TS.
 3. A method of making a recombinant mesophilic bacteriumthat is temperature-sensitive (TS), comprising: introducing into thegenome of a mesophilic bacterium a nucleic acid construct comprising aTS essential nucleic acid molecule from a psychrophilic bacterium, andfunctionally replacing the mesophilic bacterium's homolog of the TSessential nucleic acid molecule, thereby making a recombinant mesophilicbacterium that is TS, wherein a protein encoded by the TS essentialnucleic acid molecule is operable at a temperature less than 30° C. andinoperable at a temperature greater than 30° C., and wherein therecombinant mesophilic bacterium that is TS has a restrictivetemperature between 33° C. and 44° C.
 4. The method of claim 3, whereinthe psychrophilic bacterium is Colwellia sp., Pseudoalteromonas sp., orShewanella sp.
 5. The method of claim 3, wherein the protein encoded bythe TS essential nucleic acid molecule comprises the amino acid sequenceshown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or28.
 6. The method of claim 3, wherein one or more control sequences areoperably linked to the TS essential nucleic acid molecule.
 7. The methodof claim 3, wherein introducing into the genome of the mesophilicbacterium the nucleic acid construct comprising a TS essential nucleicacid molecule from the psychrophilic bacterium comprises introducinginto the genome of the mesophilic bacterium by homologous recombinationone or more TS essential nucleic acid molecules from the psychrophilicbacterium.
 8. A recombinant mesophilic bacterium that istemperature-sensitive (TS) made by the method of claim
 3. 9. Therecombinant mesophilic bacterium that is TS of claim 8, wherein therecombinant mesophilic bacterium that is TS is a Francisella novicidacell.
 10. The recombinant mesophilic bacterium that is TS of claim 8,wherein the recombinant mesophilic bacterium that is TS is afermentative microbial strain or a bioremediation strain.
 11. Therecombinant mesophilic bacterium that is TS of claim 8, wherein thepsychrophilic bacterium is Colwellia sp., Pseudoalteromonas sp., orShewanella sp.
 12. The recombinant mesophilic bacterium that is TS ofclaim 8, wherein the psychrophilic bacterium is Pseudoalteromonashaloplanktis or C. psychrerhraea.
 13. The recombinant mesophilicbacterium that is TS of claim 8, wherein the mesophilic bacterium thatis TS is Haemophilus sp., Vibrio sp., Escherichia sp., Streptococcussp., Burkholderia sp., Campylobacter sp., Neisseria sp., Francisellasp., Salmonella sp. or Mycobacterium sp.
 14. The recombinant mesophilicbacterium that is TS of claim 8, wherein the TS essential nucleic acidmolecule comprises the nucleotide sequence shown in SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, wherein expression of the TSessential nucleic acid molecule confers temperature sensitivity.
 15. Therecombinant mesophilic bacterium that is TS of claim 8, wherein theprotein encoded by the TS essential nucleic acid molecule comprises theamino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, or
 28. 16. The recombinant mesophilic bacterium that isTS of claim 8, wherein the protein encoded by the TS essential nucleicacid molecule is operable at a temperature above of 4° C. and inoperableat a temperature greater than 30° C.
 17. The recombinant mesophilicbacterium that is TS of claim 8, wherein the nucleic acid constructcomprising the TS essential nucleic acid molecule comprises a vector.18. A composition comprising the recombinant mesophilic bacterium thatis TS of claim
 8. 19. The composition of claim 18, wherein thecomposition is an immunogenic or therapeutic composition.
 20. Thecomposition of claim 18, further comprising an adjuvant.
 21. Thecomposition of claim 18, further comprising a pharmaceuticallyacceptable carrier.
 22. The composition of claim 18, comprising morethan one type of recombinant mesophilic bacterium that is TS.
 23. Thecomposition of claim 18, wherein the recombinant mesophilic bacteriumthat is TS is a Francisella sp., Salmonella sp., or Mycobacterium sp.